Human CRF2 alpha and beta splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

Corticotropin releasing factor (CRF) receptors belong to the super-family o f G protein-coupled receptors. These receptors are classified into two subt ypes (CRF1 and CRF2). Both receptors are positively coupled to adenylyl cyc lase but they have a distinct pharmacology and distribution in brain. Two i soforms belonging to the CRF2 subtype receptors, CRF2 alpha and CRF2 beta, have been identified in rat and man. The neuropeptides CRF and urocortin me diate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recent ly identified hCRF(2 beta) receptor. We have used radioligand binding with [I-125]-tyr(0)-sauvagine and a gene expression assay in which the firefly l uciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [I-125]-tyr(0)-sauvagine binding to the hCRF(2 bet a) receptor were monophasic while dissociation kinetics were biphasic, in a greement with the kinetics results obtained with the hCRF(2 alpha) receptor . Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and wi th parameters obtained with the hCRF(2 alpha) receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [I-125]-tyr(0)-s auvagine to both hCRF(2) receptor isoforms in a similar manner. The rank or der of potency of CRF agonist peptides in competition experiments was ident ical for both hCRF2 isoforms (urocortin > sauvagine > urotensin 1 > r/hCRF > alpha-helical CRF(9-41) > oCRF). Similarly, agonist potency was similar f or the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist alpha-helical CRF(9-41) exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF(2) receptor isoforms. Taken together, these results indicate that the pharmac ological profiles of the CRF2 splice variants are identical. This indicates that the region of the N-terminus that varies between the receptors is pro bably not important in the binding of peptide CRF receptor ligands or funct ional activation of the receptor. (C) 1999 Elsevier Science Ltd. All rights reserved.